Novel virus encoded chemokines determine the tissue tropism of human cytomegalovirus (HCMV)

ABSTRACT

The patent application PCT/EP02/01867 (WO 02/066629) “Recombinant vector containing infectious human cytomegalovirus genome with preserved wild-type characteristics of clinical isolates” describes the cloning of a leukotropic and endothelial cell tropic clinical isolate of human cytomegalovirus (HCMV) as a bacterial artificial chromosome (BAC) in  E. coli . The cloned wild-type genome of HCMV was designated FIX-BAC (Fusion-Inducing-Factor-X)-BAC (Hahn, Khan et al., 2002). The patent application PCT/EP02/01867 (WO 02/066629) also describes the construction of virus mutants using FIX-BAC technology and subsequent phenotypical testing of virus mutants for loss of wild-type features such as leukocyte and endothelial cell tropism. The genetic determinants of endothelial cell and leukocyte tropism were assigned to the UL132-UL128 genetic locus of HCMV. Moreover, the patent application PCT/EP02/01867 (WO 02/066629) describes novel transcripts within the UL131-UL128 genetic locus which are differentially spliced. The current patent application describes in more detail the UL131-128 transcripts of clinical isolates of HCMV. Translation of the newly identified transcripts showed novel open reading frames (orfs) coding for novel putative C×C and CC chemokines which are of crucial importance for HCMV pathogenesis and tissue tropism.

DESCRIPTION

[0001] The previous patent application PCT/EP02/01867 (WO 02/066629)describes the cloning of FIX-BAC and assigned the genetic determinantsof leukocyte and endothelial cell tropism of HCMV to the UL132-UL128genetic locus of HCMV.

[0002] In the current patent application specific virus mutantstargeting individual genes within the UL131-128 region as well as at theborders of UL131-128 were constructed using a sited-directed PCR-basedapproach in E. coli. Recombination functions of bacteriophage λ (α/β/γ)were transiently provided on a plasmid in E. coli DH10B containing atleast one copy of the FIX-BAC genome. The recombination fragment wasgenerated by PCR over a kanamycin resistance marker (pACYC177, NEBBiolabs).

[0003] The following primers were used: RVFIXΔUL130-132 (P-132-for:5′-AAA CCA CGT CCT CGT CAC ACG TCG TTC GCG GAC ATA GCA AGA; [SEQ. ID NO:1] AAT CCA CGT CGC CAC ATC TCG AGA CGA TTT ATT CAA CAA AGC CAC G-3′P-130-rev: 5′-AAC GGC GTC AGG TCT TTG GGA CTC ATG ACG CGC GGT TTT CAA;[SEQ. ID NO: 2] AAT TCC CTG CGC GCG CGA CGG GCG CCA GTG TTA CAA CCA ATTAAC C-3′) RVFIXΔUL130 (P-130-for: 5′-GCG CCA CAC GCC CGG AGC CTC GAG TTCAGC GTG CGG CTC TTT; [SEQ. ID NO: 3] GCC AAC TAG CCT GCG TCA CGG CGA TTTATT CAA CAA AGC-3′ P-130-rev: 5′-AAC GGC GTC AGG TCT TTG GGA CTC ATG ACGCGC GGT TTT CAA; [SEQ. ID NO: 4) AAT TCC CTG CGC GCG CGA CGG GCG CCA GTGTTA CAA CCA ATT AAC C-3′ RVFIXΔ128K (P-128-for-kons: 5′-TGC GTT CTG TGGTGC GTC TGG ATC TGT CTC TCG ACG TTT CTG, [SEQ. ID NO: 5] ATA GCC ATG TTCCAT CGA CGA TTT ATT CAA CAA AGC CAC G-3′ P-128-kons2: 5′-CGG CAC ACA TCCAGC CGT TTG TGT TTC TTA ACG CTC TCC AGG [SEQ. ID NO: 6]) TAC TGA TCC AGGCCC ACG GCC AGT GTT ACA ACC AAT TAA-3′ RVFIXΔUL127 (P-127-for: 5′-TTGAGA TTT CTG TCG CCG ACT AAA TTC ATG TCG CGC GAT AGT, [SEQ. ID NO: 7] GGTGTT TAT CGC CGA TAG CGA TTT ATT CAA CAA AGC CAC-G3′ P-127-rev: 5′-AATATT GAT TTA CGC TAT ATA ACC AAT GAC TAA TAT GGC TAA; [SEQ. ID NO: 8] TGGCCA ATA TTG ATG CAA GCC AGT GTT ACA ACC AAT TAA-3′) RVFIXΔUL148(P-148-for: 5′-GAC TAT GTG CAT GTT CGG CTA CTG AGC TAC CGA GGC GAC CCC,[SEQ. ID NO: 9] CTG GTC TTC AAG CAC ACT CGA TTT ATT CAA CAA AGC CAC-3′P-148-rev: 5′-CAC CAG GTA GGT TAT CAA AAC GCG AGC CCA TAT CGC CGC CAT;[SEQ. ID NO: 10) CAT TGT AAT CAG CAA TGT GCC AGT GTT ACA ACC AAT TAA-3′RVFIXΔUL132K (P-132-forK: 5′-ACG TCC TCG TCA CAC GTC GTT CGC GGA CAT AGCAAG AAA TTC, [SEQ. ID NO: 11] ACG TCG CCA CGT CTC GAG ACG ATT TAT TCAACA AAG CCA-3′ P-132-revk: 5′-AAG GTT CTT CCA TTT CCG AGG CGG TCA GTTCAT CGT ACA CCG; [SEQ. ID NO: 12]) AGA CGT AGT ACC TGA TGG GGC CAG TGTTAC AAC CAA TTA ACC-3′ RVFIXΔUL132-128 (P-131-for: 5′-TGT CTT TCG GTTCCA ACT CTT TCC CCG CCC CAT CAC CTC GCC; [SEQ. ID NO: 13] TGT ACT ATGTGT CGA TTT ATT CAA CAA AGC CAC G-3′ P-128-rev: 5′-TCG CGC GAC ATG AATTTA GTC GGC GAC AGA AAT CTC GAA ACG; [SEQ. ID NO: 14]) CGT ATT TCG GACAAA CAC ACA TGC CAG TGT TAC AAC CAA TTA ACC-3′ RVFIXΔUL133-148(P-133-for: 5′-CGC TGT AGG GAT AAA TAG TGC GAT GGC GTT TGT GGG AGA ACG,[SEQ. ID NO: 15] CAG TAG CGA TGG GTT GCG ACG TGC ACC GAT TTA TTC AAC AAAGCC ACG-3′ P-148-rev: 5′-CAC CAG GTA GGT TAT CAA AAC GCG AGC CCA TAT CGCCGC CAT; [SEQ. ID NO: 16] CAT TGT AAT CAG CAA TGT GCC AGT GTT ACA ACCAAT TAA-3′) RVFIXΔUL131K (P-131-fork: 5′-CAG TCT GCA ACA TGC GGC TGT GCTGGG TGT GGC TGT CTG TTT, [SEQ. ID NO: 17] GTC TGT GCG CCG TGG TGC CGATTT ATT CAA CAA AGC CAC-3′ P-131-rev: 5′-GCT AGT TGG CAA AGA GCC GCA CGCTGA ACT CGA GGC TCC GGG; [SEQ. ID NO: 18] CGT GTG GCG GCC AGT GTT ACAACC AAT TAA CC-3′) RVFIXΔ146-147 (P-UL146-for: 5′-GAT TTT CCG GGA ATACCG GAT ATT ACG AAT TAC TGG TAG TGA, [SEQ. ID NO: 19] CGT AGA TAA TAAAAT TAT ACG ATT TAT TCA ACA AAG CCA CG-3′ P-UL147-rev: 5′-CAC CAA AGCCGT TAG CGT GCC CAG AGC TAC CGC ACG GTA AAA. [SEQ. ID NO: 20] TAG GGACAT GAG CCA GTG TTA CAA CCA ATT AAC C-3′)

[0004] The Southern Blot was probed with the kanamycin specific probepAcyc. The generation and testing of the mutants is described in thetext. M: molecualr weight marker 1 kb ladder.

[0005] Tabel 1 summarizes the phenotypical testing of RVFIX virusmutants. TABLE 1 Leukocyte, Monocyte and HUVEC tropism PMNL-TropismMO-Tropism HUVEC-Tropism RVFIX WT positive positive HUVEC+ RVFIX_(Δ)UL130 negative negative HUVEC− RVFIX _(Δ)UL131 negative negativeHUVEC− RVFIX _(Δ)UL132 positive positive HUVEC+ RVFIX _(Δ)UL128 negativenegative HUVEC− RVFIX_(Δ)UL131-128 negative negative HUVEC−RVFIX_(Δ)UL127 positive positive HUVEC+ RVFIX_(Δ)UL148 positive positiveHUVEC+ RVFIX_(Δ)UL133-148 weak pos. positive HUVEC+ RVFIX_(Δ)UL146-147weak pos. positive HUVEC+

[0006] Only mutants with a deletion of either individual genes UL131,UL130, UL128 or the entire UL131-128 locus lost tropism for bothendothelial cells and leukocytes. When genes next to UL131-128 weredeleted (UL132, UL127 or UL148) the virus mutants were perfectly capableof infecting endothelial cells and leukocytes confirming the UL131-128locus as the genetic determinant of endothelial cell and leukocytetropism. When a deletion between UL133-148 was introduced into the RVFIXgenome the resulting virus RVFIXΔUL133-148 retained the capability toinfect endothelial cells and leukocytes, however a predominant monocytetropism was observed. Since it is known from literature (Penfold,Dairaghi et al., 1999; Mocarski, Jr., 2002) that UL146 is a functionalC×C chemokine, the predominant monocyte tropism of the virus mutantRVFIXΔUL133-148 was attributed to the loss of UL146. In order to confirmthis notion, a virus mutant was constructed which UL146-UL147 wasdeleted. The virus mutant RVFIXΔ146-147 confirmed the RVFIXΔUL133-148phenotype.

[0007] The role of leukocyte attraction in cytomegalovirus infection wasrecently underscored by the finding that, in MCMV, two differentiallyspliced CC chemokine homologs (Fleming, Davis-Poynter et al., 1999;MacDonald, Li et al., 1997; MacDonald, Burney et al., 1999) encoded bym131-129 (MCK-1 and MCK-2) are important determinants of virusdissemination (MacDonald, Li, & Virgin, 1997; MacDonald, Burney,Resnick, & Virgin, IV, 1999; Saederup, Lin et al., 1999; Saederup,Aguirre et al., 2001), acting as proinflammatory signals that recruitleukocytes to the site of infection to increase virus spreading. Therelevance of chemoattraction is highlighted in this study by thephenotype of mutants RVΔUL146-147 and RVΔUL133-148, both bearing afunctional UL131-128 locus while lacking the viral C×C chemokine genesUL146-147. UL146 product is a potent attractor and activator of humanPMNL in vitro (Penfold, Dairaghi, Duke, Saederup, Mocarski, Kemble, &Schall, 1999). The mentioned virus mutants RVΔUL146-147 and RVΔUL133-148share a full endothelial cell and monocyte tropism, but are onlyinefficiently transmitted to PMNL (polymorphonuclear leukocytes). Theseresults suggest that genes within UL131-128 are sufficient both forlocal endothelial spread and for attraction of (and virus passage to)monocytes, while the additional chemotactic factors encoded by UL146-147enhance virus dissemination by attracting PMNL and favouring theirinfection. The chemotactic activity of each individual gene product ofthe UL131-128 locus, as well as the cooperation with other viral orcellular gene signalling molecules, remain to be further elucidated.

[0008] As described in patent application PCT/EP02/01867 (WO 02/066629)5′ and 3′ RACE analyses had led to the identification of novel viraltranscripts running through the genetic locus UL131-UL128 with a startcodon at the beginning of UL131 and a stop codon and poly A signal atthe end of UL128. The current patent application provides a more indepth analysis of the newly identified transcripts on thetranscriptional and translational level. FIG. 4A-C showes a sequencecomparison of the newly identified RACE clones 95-3, 95-8 and 95-11 toFIX-BAC genomic sequence. FIG. 5 shows the entire UL131-128 geneticlocus and predicted individual genes.

[0009] Transcript analysis was performed by rapid amplification of cDNAends (RACE). First strand cDNA synthesis and rapid amplification of cDNAends (RACE) was performed using the SMART™ RACE cDNA amplification kit(Clontech) according to the manufacturer's instructions. RACE productswere cloned into the pT-Adv vector (Clontech) using the AdvanTAge™ PCRcloning kit for analysis and sequencing. For rapid ampification of cDNAends (RACE) from the 5′ RACE cDNA sample the following primers wereused: primer 57-GSP1: 5′-CGG CAC ACA TCC AGC CGT TTG TGT TTC TTA 3′[SEQ. ID NO: 35]; primer 72-GSP1: 5′-TAA CGC TCT CCA GGT ACT GAT CCA GGCCCA-3′ [SEQ. ID NO: 36]; primer 73-GSP1: 5′-TCG TCA GTT TGT TGT GTA CGACCT GGC GTG-3′ [SEQ. ID NO: 37]; primer 74-GSP1: 5′-TAT TGG CCT CGG TGAACG TCA ATC GCA CCT -3′ [SEQ. ID NO: 38]. For rapid ampification of cDNAends from the 3′ RACE cDNA sample the following primers were used:primer 56-GSP2: 5′-TGT GTC GGG TGT GGC TGT CTG TTT GTC TGT-3′ [SEQ. IDNO: 39]; primer 75-GSP2: 5′-TCT GCT TCG TCA CCA CTT TCA CTG CCT GCT-3′[SEQ. ID NO: 40]; primer 76-GSP2: 5′-CGC AGA AGA ATG TTG CGA ATT CAT AAACGT-3′ [SEQ. ID NO: 41]; primer 77-GSP2: 5′-GCT GCG GTG TCC GGA CGG CGAAGT CTG CTA-3′ [SEQ. ID NO: 42]; primer 78-GSP2: 5′-CCA GCT GGC AGA TTCCCA AAC TAA TGA AAG-3′ [SEQ. ID NO: 43]; primer 93-GSP2: 5′-CTT TCG GTTCCA ACT CTT TCC CCG CCC CAT-3′ [SEQ. ID NO: 44]; primer 94-GSP2: 5° CACCTC GCC TAT ACT ATG TGT ATG ATG TCT-3′ [SEQ. ID NO: 45]; primer 95-GSP2:5′-CTC TCT TTC TCA GTC TGC AAC ATG CGG CTG-3′ [SEQ. ID NO: 46]; primer96-GSP2: 5′-GTT GTC CAA GCC GTC GCT CGC ATC GTA GTG-3′ [SEQ. ID NO: 47];primer 97-GSP2: 5′-CAT AAT AAA GCT CTC TTT CTC AGT CTG CAA-3′ [SEQ. IDNO: 48]; primer 98-GSP2: 5′-TAT GAT GTC TCA TAA TAA AGC TCT CTT TCT-3′[SEQ. ID NO: 49].

[0010] Identification of Novel Tropism Determining Transcripts

[0011] The novel and previously unrecognized transcripts running throughthe entire UL131-128 region with a predicted UL131 start codon (nt176825-176823 according to (Chee, Bankier, Beck, Bohni, Brown, Cerny,Horsnell, Hutchison, 111, Kouzarides, Martignetti, & ., 1990), and aUL128 stop codon (nt 174865-174863) were identified. The newlyidentified transcripts show a splicing event between UL128×2 and UL128×3(nt 175201-175081), either exclusively or in conjunction with splicingbetween UL131×1 and UL131×2 (nt 176589-176480). An additional splicingevent between UL128×1 and UL128×2 (nt 175459-175335) was observed inseveral clones.

[0012] 3′ RACE analysis consistently identified a single polyA stretch14±1 nucleotides 3′ to the canonical AATAAA polyA signal immediatelydownstream of the UL128 stop codon.

[0013] As depicted in FIG. 6 [HCK-1:nucleic acid:SEQ. ID NO: 50; aminoacid: SEQ. ID NO. 51, HCK-2:nucleic acid: SEQ. ID NO: 52; amino acid:SEQ. ID NO: 53] and translation of RACE clone 95-3 (FIG. 9) [FIX:nucleicacid:SEQ. ID NO: 54; 95-3:nucleic acid:SEQ. ID NO: 55; HCK-1 nucleicacid:SEQ. ID NO: 56;amino acid:SEQ. ID NO: 57] and RACE clone 95-8 (FIG.10) [FIX: nucleic acid:SEQ. ID NO: 58; 95-8:nucleic acid:SEQ. ID NO: 58;HCK-2:nucleic acid:SEQ. ID NO: 60; amino acid:SEQ. ID NO: 61] showspredicted proteins with a C×C motif (red) which is characteristic forC×C chemokines. RACE clone 95-3 encodes a 129 aa protein designatedHCK-1 (pUL131; about 15 kDA) and RACE clone 95-8 encodes a 79 aa proteindesignated HCK-2 (pUL131×1; about 9 kDA). Both proteins show a number ofN-linked glycosilation sides.

[0014] Both newly indentified transcripts (RACE clone 95-3 and RACEclone 95-8) have a splice between UL128×1 and UL128×2. RACE clone 95-3has an additional splice between UL131×1 and UL131×2 which is absent inRACE clone 95-8. Sequence comparsion between the RACE clones, FIX-BACand AD169 genomic sequences revealed that a stretch of 7×nt A (blue) inUL131 of FIX-BAC and the RACE clones 95-3 and 95-8 is extended to astretch of 8×nt A (blue) in AD169 genomic sequence. Translation of theRACE clones 95-3 (FIG. 9) and 95-8 (FIG. 10) showed an open readingframe (ORF) with a conserved C×C C C chemokine motif (red) in FIX-BACwhich is destroyed by the extension of 7×nt A to 8×nt A in thefibroblast adapted laboratory strain AD169. In RACE clone 95-3 thesplice between UL131×1 and UL131×2 removes the stop codon at the end ofUL131×1 and codes for a putative 129 aa C×C chemokine like protein(designated HCK-1; Human cytomegalovirus chemokine like protein),whereas in RACE clone 95-8 the stop codon at the end of UL131×1 is usedto truncate the C×C chemokine like protein HCK-2 to 79 aa.

[0015] Putative CC chemokine like proteins designated HCK-3 and HCK-4are encoded by the predicted UL128 ORF (FIG. 7) [HCK-3:nucleic acid:SEQ.ID NO: 62; amino acid:SEQ. ID NO: 63; HCK-4:nucleic acid:SEQ. ID NO: 64;amino acid:SEQ. ID NO: 65]. However, RACE analyses have not yet fullyconfirmed the 5′ start of these transcripts. HCK-3 (pUL128×1) (FIG. 12)[FIX:nucleic acid:SEQ. ID NO: 66; 128B:nucleic acid:SEQ. ID NO: 67;HCK-3:nucleic acid:SEQ. ID NO: 68; amino acid:SEQ. ID NO: 69]is a 59 aaprotein (about 7 kDA) and HCK-4 (pUL128) (FIG. 13) [FIX:nucleicacid:SEQ. ID NO: 70; 128A:nucleic acid:SEQ. ID NO: 71; HCK-4:nucleicacid:SEQ. ID NO: 72; amino acid:SEQ. ID NO: 73] is a 171 aa protein(about 20 kDA). Splicing events between UL128×1, UL128×2 and UL128×3fuse the three UL128 exons to form the 171 aa protein HCK-4. In case ofHCK-3 the splice between UL128×1 and UL128×2 is absent and thus the TGAstop codon at the end of UL128×1 truncates the protein HCK-3 to 59 aa.

[0016] RACE clone 95-11 (FIG. 11) [FIX:SEQ. ID NO: 74; 95-11:S EQ. IDNO: 75] confirms that UL128 is composed of three exons.

[0017] Interestingly, in RACE clone 95-11 the stretch of 7×nt A (presentin RACE clones 95-3 and 95-8) is extended to a stretch of 9×nt A. Thisextension from 7× to 9×nt A destroys the C×C motif in RACE clone 95-11and takes the predicted HCK-1 and HCK-2 proteins out of frame. It can bespeculated that by increasing or decreasing the numbers of nt (forexample nt A, nt T, nt G or nt C) in a given transcript, HCMV has founda way of transcriptional regulation of tissue tropism. It can bepictured that in a given cell type (for example fibroblasts)predominantly one transcript (for example 95-11) is synthesized overothers (for example 95-8 or 95-3), thereby introducing a frame shift inan open reading frame (for example HCK-1 and HCK-2) whose product isnecessary for tissue specific infection. It can also be pictured that inother cells for example endothelial cells, monocytes, leukocytes,dendritic cells or progenitor cells predominantly those transcripts aremade (for example 95-3, 95-8, 128A and 128B) whose protein products (forexample HCK-1, HCK-2, HCK-3 and HCK-4) encode viral chemokines andmicrofusion inducing factors (for example UL130, HCK-5) (FIG. 14)[nucleic acid:SEQ. ID NO: 76; amino acid:SEQ. ID NO: 77].

[0018] Closer analysis of the newly encoded proteins HCK-1 and HCK-2shows that they have N-linked gylcosilation sites. It can be speculatedthat HCK-1 may be membrane bound and is trafficking through theendoplasmatic reticulum. This membrane bound HCK-1 could be of crucialimportance for inducing the microfusion event (in conjunction withpUL130, HCK-5) between endothelial cells and leukocytes, monocytes,makrophages and dendritic cells or possibly other cell types. It can beassumed that HCK-2 may be a soluble chemokine which would be necessaryto attract leukocytes (in conjunction with the C×C chemokines UL146 andUL147) to the site of infection. Thus HCK-1 and HCK-2 are majorpathogenicity factors for virus dissemination in vivo and in vitro. Itcan further be assumed that the newly identified CC chemokines HCK-3 andHCK-4 may attract monocytes, macrophages, dendritic cells and possiblyhematopoietic progenitor cells or stem cells to the site of infectionand that concomitantly the infectious virus is spread via microfusionpossibly by the use of HCK-1 and HCK-5 protein products. It can bepictured that chemokine receptors interact with the HCK-1, HCK-2, HCK-3,HCK-4 and HCK-5 protein and that the microfusion event occurs viareceptor internalisation. Adhesion molecules which are upregulated byHCMV infection may provide assistance in the attachment, recruitment andmicrofusion process.

[0019] Transcript Analyses by Northern Blot

[0020] Northern blots from fibroblasts infected with RVFIX (FIG. 8),when hybridized with a UL131-128 specific probe, showed an upper(2.0-1.8 kb) band and a lower (0.8-0.7 kb) band. The 0.8-0.7 kb band canbe interpreted as a UL128-specific transcript, the 2.0-1.8 kb band asthe long UL131-128 encompassing transcript. Independent promoters maydrive each transcript, one predicted upstream of the UL131 gene and theother within the UL131-130 region. The lack of a UL130 specifictranscript suggests that the UL130 protein may be translated from thepolycistronic 2.0-1.8 kb mRNAs through either translational reinitiationor an IRES-like mechanism.

[0021] Northern analyses of RVFIXΔUL130 or RVFIXΔUL131K infectedfibroblasts shows that following the kan^(R) cassette insertion intoeither UL131 or UL130 the stability of all transcripts of the UL131-128region was altered (2.0-1.8 kb transcript was absent and 0.8-0.7 kbtranscript was strongly diminished), whereas kan^(R) insertion intoRVFIXΔUL128 shifted both transcripts (FIG. 8). In Toledo infected cellsboth mRNA bands are missing as a consequence of truncation of UL128 anddislocation of the polyA signal (FIG. 8). Taken together, these datasuggest that a targeted deletion of individual genes within UL131 toUL128 affects the stability of transcripts of the entire locus.Functionally, the lack of endothelial cell and leukocyte tropismobserved in Toledo may correlate with the failure to express allUL131-128 transcripts. In contrast, point mutations in the UL131-128region of the laboratory strain AD169 as compared to RVFIX neitheraffect mRNA mobility nor stability. However, in AD169 a stretch of 7×ntA in RVFIX is extended to 8×nt A in laboratory strain AD169. As aresult, the ORF of pUL131 (HCK-1) and pUL131×1 (HCK-2) in AD169 isframeshifted.

[0022] By construction of individual virus mutants which deleteindividual genes UL131, UL130, UL128 it could be demonstrated that HCMVloses its tropism for monocytes, leukocytes and endothelial cells whenthe genetic region coding for the viral proteins HCK-1, HCK-2, HCK-3,HCK-4 or HCK-5 is removed from the virus genome. It is reasonable toassume that infection of cell types such as dendritic cells,macrophages, progenitor cells, B- and T-lymphocytes, hematopietic stemcells may occur in the same fashion and that the UL131-128 locus of HCMVand its encoded protein products described are indispensable for theinfection process in vivo and in vitro.

[0023] Rescue of Leukotropism and Endothelial Cell Tropism by EctopicReinsertion of the ULI31-128 Region of FIX-BAC into the LaboratoryStrain AD169.

[0024] In order to show that the UL131-128 region of FIX-BAC issufficient to rescue tissue tropism of a an endothelial cell tropism andleukotropism incompetent strain, the UL131-128 genetic locus of FIX-BACwas cloned into the vector pOR16K-zeo next to an FRT site. In AD169-BACthe UL40 region was deleted and replaced with a kanamycin cassetteflanked by FRT sites (from plasmid pcp015). Subsequently the kanamycinresistance marker was removed by flip recombinase (provided by plasmidpcp20) in E. coli. Ectopic insertion of the UL131-128 region fromFIX-BAC (cloned into pORI6K-zeo) into AD169ΔUL40FRT-BAC was achieved byflip recombinase in E. coli and selection for kan^(R) and zeo^(R)resistant clones. The reconstituted virus RVAD169ΔUL40+UL131-128 couldinfect endothelial cells and leukocytes. However, the leukocyte tropismwas predominantly restricted to monocytes, as expected in a virus whichlacks the C×C chemokine coding genes UL146 and UL147.

[0025] The genes UL131, UL130, UL128 and the protein products with novelstructure encoded (namely HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5) are offundamental importance for infection, dissemination and spread of HCMVin the human body. It can be pictured that these genes and proteins arekey players in disease conditions such as vascular disease andatherosclerosis development. They are targets for drug design (smallmolecules, anti-sense RNA, siRNAs), anti-viral chemotherapy, vaccinedevelopment and gene therapy against HCMV and other virus induceddiseases (for example HIV), as well as diseases such as cancer,autoimmune disorders and atherosclerosis. Since viruses like HCMVco-evolved with the host during human evolution it, is reasonable toassume that the newly described C×C and CC chemokines of novel structuremay have as yet unidentified chemokine homologues in humans (for examplesecreted by human immune cells).

[0026] Reference List

[0027] Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M.,Cerny, R., Horsnell, T., Hutchison, C. A., III, Kouzarides, T.,Martignetti, J. A., and . (1990). Analysis of the protein-coding contentof the sequence of human cytomegalovirus strain AD169. Curr TopMicrobiol Immunol 154, 125-169.

[0028] Fleming, P., Davis-Poynter, N., Degli-Esposti, M., Densley, E.,Papadimitriou, J., Shellam, G., and Farrell, H. (1999). The murinecytomegalovirus chemokine homolog, m131/129, is a determinant of viralpathogenicity. J Virol 73, 6800-6809.

[0029] Hahn, G., Khan, H., Baldanti, F., Koszinowski, U. H., Revello, M.G., and Gerna, G. (2002). The human cytomegalovirus ribonucleotidereductase homolog UL45 is dispensable for growth in endothelial cells,as determined by a BAC-cloned clinical isolate of human cytomegaloviruswith preserved wild-type characteristics. J Virol 76, 9551-9555.

[0030] MacDonald, M. R., Burney, M. W., Resnick, S. B., and Virgin, H.W., IV (1999). Spliced mRNA encoding the murine cytomegaloviruschemokine homolog predicts a beta chemokine of novel structure. J Virol73, 3682-3691.

[0031] MacDonald, M. R., Li, X. Y., and Virgin, H. W. (1997). Lateexpression of a beta chemokine homolog by murine cytomegalovirus. JVirol 71, 1671-1678.

[0032] Mocarski, E. S., Jr. (2002). Immunomodulation bycytomegaloviruses: manipulative strategies beyond evasion. TrendsMicrobiol 10, 332-339.

[0033] Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N.,Mocarski, E. S., Kemble, G. W., and Schall, T. J. (1999).Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci U SA 96, 9839-9844.

[0034] Saederup, N., Aguirre, S. A., Sparer, T. E., Bouley, D. M., andMocarski, E. S. (2001). Murine cytomegalovirus CC chemokine homologMCK-2 (m131-129) is a determinant of dissemination that increasesinflammation at initial sites of infection. J Virol 75, 9966-9976.

[0035] Saederup, N., Lin, Y. C., Dairaghi, D. J., Schall, T. J., andMocarski, E. S. (1999). Cytomegalovirus-encoded beta chemokine promotesmonocyte-associated viremia in the host. Proc Natl Acad Sci U S A 96,10881-10886.

1 79 1 88 DNA Artificial Sequence oligonucleotide primer 1 aaaccacgtcctcgtcacac gtcgttcgcg gacatagcaa gaaatccacg tcgccacatc 60 tcgagacgatttattcaaca aagccacg 88 2 88 DNA Artificial Sequence oligonucleotideprimer 2 aacggcgtca ggtctttggg actcatgacg cgcggttttc aaaattccctgcgcgcgcga 60 cgggcgccag tgttacaacc aattaacc 88 3 81 DNA ArtificialSequence oligonucleotide primer 3 gcgccacacg cccggagcct cgagttcagcgtgcggctct ttgccaacta gcctgcgtca 60 cggcgattta ttcaacaaag c 81 4 88 DNAArtificial Sequence oligonucleotide primer 4 aacggcgtca ggtctttgggactcatgacg cgcggttttc aaaattccct gcgcgcgcga 60 cgggcgccag tgttacaaccaattaacc 88 5 82 DNA Artificial Sequence oligonucleotide primer 5tgcgttctgt ggtgcgtctg gatctgtctc tcgacgtttc tgatagccat gttccatcga 60cgatttattc aacaaagcca cg 82 6 81 DNA Artificial Sequence oligonucleotideprimer 6 cggcacacat ccagccgttt gtgtttctta acgctctcca ggtactgatccaggcccacg 60 gccagtgtta caaccaatta a 81 7 81 DNA Artificial Sequenceoligonucleotide primer 7 ttgagatttc tgtcgccgac taaattcatg tcgcgcgatagtggtgttta tcgccgatag 60 cgatttattc aacaaagcca c 81 8 81 DNA ArtificialSequence oligonucleotide primer 8 aatattgatt tacgctatat aaccaatgactaatatggct aatggccaat attgatgcaa 60 gccagtgtta caaccaatta a 81 9 81 DNAArtificial Sequence oligonucleotide primer 9 gactatgtgc atgttcggctactgagctac cgaggcgacc ccctggtctt caagcacact 60 cgatttattc aacaaagcca c81 10 81 DNA Artificial Sequence oligonucleotide primer 10 caccaggtaggttatcaaaa cgcgagccca tatcgccgcc atcattgtaa tcagcaatgt 60 gccagtgttacaaccaatta a 81 11 81 DNA Artificial Sequence oligonucleotide primer 11acgtcctcgt cacacgtcgt tcgcggacat agcaagaaat tcacgtcgcc acgtctcgag 60acgatttatt caacaaagcc a 81 12 84 DNA Artificial Sequence oligonucleotideprimer 12 aaggttcttc catttccgag gcggtcagtt catcgtacac cgagacgtagtacctgatgg 60 ggccagtgtt acaaccaatt aacc 84 13 76 DNA ArtificialSequence oligonucleotide primer 13 tgtctttcgg ttccaactct ttccccgccccatcacctcg cctgtactat gtgtcgattt 60 attcaacaaa gccacg 76 14 87 DNAArtificial Sequence oligonucleotide primer 14 tcgcgcgaca tgaatttagtcggcgacaga aatctcgaaa cgcgtatttc ggacaaacac 60 acatgccagt gttacaaccaattaacc 87 15 90 DNA Artificial Sequence oligonucleotide primer 15cgctgtaggg ataaatagtg cgatggcgtt tgtgggagaa cgcagtagcg atgggttgcg 60acgtgcaccg atttattcaa caaagccacg 90 16 81 DNA Artificial Sequenceoligonucleotide primer 16 caccaggtag gttatcaaaa cgcgagccca tatcgccgccatcattgtaa tcagcaatgt 60 gccagtgtta caaccaatta a 81 17 81 DNA ArtificialSequence oligonucleotide primer 17 cagtctgcaa catgcggctg tgctgggtgtggctgtctgt ttgtctgtgc gccgtggtgc 60 cgatttattc aacaaagcca c 81 18 74 DNAArtificial Sequence oligonucleotide primer 18 gctagttggc aaagagccgcacgctgaact cgaggctccg ggcgtgtggc ggccagtgtt 60 acaaccaatt aacc 74 19 83DNA Artificial Sequence oligonucleotide primer 19 gattttccgg gaataccggatattacgaat tactggtagt gacgtagata ataaaattat 60 acgatttatt caacaaagcc acg83 20 76 DNA Artificial Sequence oligonucleotide primer 20 caccaaagccgttagcgtgc ccagagctac cgcacggtaa aatagggaca tgagccagtg 60 ttacaaccaattaacc 76 21 408 DNA Human cytomegalovirus 21 gtctgcaaca tgcggctgtctcgggtgtgg ctgtctgttt gtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagaccgcagaaaaa aacgattatt accgagtacc gcattactgg 120 gacgcgtgct ctcgcgcgctgcctgaccaa acccgttaca agtatgtgga acagctcgtg 180 gacctcacgt tgaactaccactacgatgcg agccacggct tggacaactt tgacgtgctc 240 aagagaatca acgtgaccgaggtgtcgttg ctcatcagcg actttatacg tcagaaccgt 300 cgcggcggca ccaacaaaaggaccacgttc aacgccgccg gttcgctggc gcctcacgcc 360 cggagcctcg agttcagcgtgcggctcttt gccaactagc ctgcgtca 408 22 516 DNA Human cytomegalovirus 22gtctgcaaca tgcggctgtg tcgggtgtgg ctgtctgttt gtctgtgcgc cgtggtgctg 60ggtcagtgcc agcgggagac cgcagaaaaa aacgattatt accgagtacc gcattactgg 120gacgcgtgct ctcgcgcgct gcctgaccaa acccgttaca agtatgtgga acagctcgtg 180gacctcacgt tgaactacca ctacgatgcg agccacggct tggacaactt tgacgtgctc 240aagaggtgag ggtacgcgct aaaggtgtat gacaacggga aggtaagggc gaacgggtaa 300cgggtaggta accgcatggg gtgtgaaatg acgttcggaa cctgtgcttg cagaatcaac 360gtgaccgagg tgtcgttgct catcagcgac tttagacgtc agaaccgtcg cggcggcacc 420aacaaaagga ccacgttcaa cgccgccggt tcgctggcgc ctcacgcccg gagcctcgag 480ttcagcgtgc ggctctttgc caactagcct gcgtca 516 23 410 DNA Humancytomegalovirus 23 gtctgcaaca tgcggctgtg tcgggtgtgg ctgtctgtttgtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagac cgcagaaaaa aaaacgattattaccgagta ccgcattact 120 gggacgcgtg ctctcgcgcg ctgcctgacc aaacccgttacaagtatgtg gaacagctcg 180 tggacctcac gttgaactac cactacgatg cgagccacggcttggacaac tttgacgtgc 240 tcaagagaat caacgtgacc gaggtgtcgt tgctcatcagcgactttaga cgtcagaacc 300 gtcgcggcgg caccaacaaa aggaccacgt tcaacgccgccggttcgctg gcgcctcacg 360 cccggagcct cgagttcagc gtgcggctct ttgccaactagcctgcgtca 410 24 516 DNA Human cytomegalovirus 24 gtctgcaaca tgcggctgtgtcgggtgtgg ctgtctgttt gtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagaccgcagaaaaa aacgattatt accgagtacc gcattactgg 120 gacgcgtgct ctcgcgcgctgcctgaccaa acccgttaca agtatgtgga acagctcgtg 180 gacctcacgt tgaactaccactacgatgcg agccacggct tggacaactt tgacgtgctc 240 aagaggtgag ggtacgcgctaaaggtgtat gacaacggga aggtaagggc gaacgggtaa 300 cgggtaggta accgcatggggtgtgaaatg acgttcggaa cctgtgcttg cagaatcaac 360 gtgaccgagg tgtcgttgctcatcagcgac tttagacgtc agaaccgtcg cggcggcacc 420 aacaaaagga ccacgttcaacgccgccggt tcgctggcgc ctcacgcccg gagcctcgag 480 ttcagcgtgc ggctctttgccaactagcct gcgtca 516 25 656 DNA Human cytomegalovirus 25 ccgtgcgtcatgagtcccaa aaacctgacg ccgttcttga cggcgttgtg gctgctattg 60 ggtcacagccgcgtgccgcg ggtacgcgca gaagaatgtt gcgaattcat aaacgtcaac 120 cacccgccggaacgctgtta cgatttcaaa atgtgcaatc gcttcaccgt cgcgtacgta 180 ttttcatgattgtctgcgtt ctgtggtgcg tctggatctg tctctcgacg tttctgatag 240 ccatgttccatcgacgatcc tcgggaatgc cagagtagat tttcatgaat ccacaggctg 300 cggtgtccggacggcgaagt ctgctacagt cccgagaaaa cggctgagat tcgcgggatc 360 gtcaccaccatgacccattc attgacacgc caggtcgtac acaacaaact gacgaactgc 420 aactacaatccgttatacct cgaagctgac gggcgaatac gctgcggcaa agtgaacgac 480 aaggcgcagtacctgctggg cgccgctggc agcgttccct atcgatggat caacctggaa 540 tacgacaagataacccggat cgtgggcctg gatcagtacc tggagagcgt taagaaacac 600 aaacggctggatgtgtgccg cgctaaaatg ggctatatgc tgcagtgaat aataaa 656 26 656 DNA Humancytomegalovirus 26 ccgcgcgtca tgagtcccaa aaacctgacg ccgttcttgacggcgttgtg gctgctattg 60 ggtcacagcc gcgtgccgcg ggtacgcgca gaagaatgttgcgaattcat aaacgtcaac 120 cacccgccgg aacgctgtta cgatttcaaa atgtgcaatcgcttcaccgt cgcgtacgta 180 ttttcatgat tgtctgcgtt ctgtggtgcg tctggatctgtctctcgacg tttctgatag 240 ccatgttcca tcgacgatcc tcgggaatgc cagagtagattttcatgaat ccacaggctg 300 cggtgtccgg acggcgaagt ctgctacagt cccgagaaaacggctgagat tcgcgggatc 360 gtcaccacca tgacccattc attgacacgc caggtcgtacacaacaaact gacgagctgc 420 aactacaatc cgttatacct cgaagctgac gggcgaatacgctgcggcaa agtgaacgac 480 aaggcgcagt acctgctggg cgccgctggc ggcgttccctatcgatggat caacctggaa 540 tacgacaaga tagcccggat cgtgggcctg gatcagtacctggagagcgt taagaaacac 600 aaacggctgg atgtgtgccg cgctaaaatg ggctatatgctgcagtgaat aataaa 656 27 533 DNA Human cytomegalovirus 27 ccgcgcgtcatgagtcccaa aaacctgacg ccgttcttga cggcgttgtg gctgctattg 60 ggtcacagccgcgtgccgcg ggtacgcgca gaagaatgtt gcgaattcat aaacgtcaac 120 cacccgccggaacgctgtta cgatttcaaa atgtgcaatc gcttcaccgt cgcgctgcgg 180 tgtccggacggcgaagtctg ctacagtccc gagaaaacgg ctgagattcg cgggatcgtc 240 accaccatgacccattcatt gacacgccag gtcgtacaca acaaactgac gagctgcaac 300 tacaatctgttatacctcga agctgacggg cgaatacgct gcggcaaagt gaacgacaag 360 gcgcagtacctgctgggcgc cgctggcagc gttccctatc gatggatcaa cctggaatac 420 gacaagataacccggatcgt gggcctggat cagtacctgg agagcgttaa gaaatacaaa 480 cggctggatgtgtgccgcgc taaaatgggc tatatgctgc agtgaataat aaa 533 28 775 DNA Humancytomegalovirus 28 ccgcgcgtca tgagtcccaa aaacctgacg ccgttcttgacggcgttgtg gctgctattg 60 ggtcacagcc gcgtgccgcg ggtacgcgca gaagaatgttgcgaattcat aaacgtcaac 120 cacccgccgg aacgctgtta cgatttcaaa atgtgcaatcgcttcaccgt cgcgtacgta 180 tttttatgat tgtctgcgtt ctgtggtgcg tctggatttgtctctcgacg tttctgatag 240 ccatgttcca tcgacgatcc tcgggaatgc cagagtagattttcatgaat ccacaggctg 300 cggtgtccgg acggcgaagt ctgctacagt cccgagaaaacggctgagat tcgcgggatc 360 gtcaccacca tgacccattc attgacacgc caggtcgtacacaacaaact gacgagctgc 420 aactacaatc cgtaagtctc ttcctcgagg gccttacagcctatgggaaa gtaagacaga 480 gggacaaaac atcattaaaa aaaaagtcta atttcacgttttgtaccccc ccttcccctc 540 cgtgttgtag gttatacctc gaagctgacg ggcgaatacgctgcggcaaa gtgaacgaca 600 aggcgcagta cctgctgggc gccgctggca gcgttccctatcgatggatc aacctggaat 660 acgacaagat aacccggatc gtgggcctgg atcagtacctggagagcgtt aagaaacaca 720 aacggctgga tgtgtgccgc gctaaaatgg gctatatgctgcagtgaata ataaa 775 29 60 DNA Human cytomegalovirus 29 cgctaaaatgggctatatgc tgcagtgaat aataaaatgt gtgtttgtcc gcaaaaaaaa 60 30 60 DNAHuman cytomegalovirus 30 cgctaaaatg ggctatatgc tgcagtgaat aataaaatgtgtgtttgtcc aaaaaaaaaa 60 31 60 DNA Human cytomegalovirus 31 cgctaaaatgggctatatgc tgcagtgaat aataaaatgt gtgtttgtcc aaaaaaaaaa 60 32 52 DNAHuman cytomegalovirus 32 cgctaaaatg ggctatatgc tgcagtgaat aataaaatgtgtgtttgtcc ga 52 33 1977 DNA Human cytomegalovirus 33 gtctgcaacatgcggctgtg tcgggtgtgg ctgtctgttt gtctgtgcgc cgtggtgctg 60 ggtcagtgccagcgggagac cgcagaaaaa aacgattatt accgagtacc gcattactgg 120 gacgcgtgctctcgcgcgct gcctgaccaa acccgttaca agtatgtgga acagctcgtg 180 gacctcacgttgaactacca ctacgatgcg agccacggct tggacaactt tgacgtgctc 240 aagaggtgagggtacgcgct aaaggtgtat gacaacggga aggtaagggc gaacgggtaa 300 cgggtaggtaaccgcatggg gtgtgaaatg acgttcggaa cctgtgcttg cagaatcaac 360 gtgaccgaggtgtcgttgct catcagcgac tttagacgtc agaaccgtcg cggcggcacc 420 aacaaaaggaccacgttcaa cgccgccggt tcgctggcgc ctcacgcccg gagcctcgag 480 ttcagcgtgcggctctttgc caactagcct gcgtcacggg aaataatatg ctacggcttc 540 tgcttcgtcaccactttcac tgcctgcttc tgtgcgcggt ttgggcaacg ccctgtctgg 600 cgtctccgtggttcacgcta acggcgaacc agaatccgtc cccgccatgg tctaaactga 660 cgtatcccaaaccgcatgac gcggcgacgt tttactgtcc ttttctctat ccctcgcccc 720 cacggtccccctcgcaattc ccggggttcc agcgggtatc aacgggtccc gagtgtcgca 780 acgagaccctgtatctgctg tacaaccggg aaggccagac cttggtggag agaagctcca 840 cctgggtgaaaaaggtgatc tggtatctga gcggtcgcaa tcagaccatc ctccaacgga 900 tgccccgaacggcttcgaaa ccgagcgacg gaaacgtgca gatcagcgtg gaagacgcca 960 agatttttggagcgcacatg gtgcccaagc agaccaagct gctacgtttc gtcgtcaacg 1020 atggcacacgttatcagatg tgtgtgatga aactggagag ctgggcccac gtcttccggg 1080 actacagcgtgtcttttcag gtgcgattga cgttcaccga ggccaataac cagacttaca 1140 ccttctgcacccatcccaat ctcatcgttt gagcccgtcg cgcgcgcagg gaattttgaa 1200 aaccgcgcgtcatgagtccc aaaaacctga cgccgttctt gacggcgttg tggctgctat 1260 tgggtcacagccgcgtgccg cgggtacgcg cagaagaatg ttgcgaattc ataaacgtca 1320 accacccgccggaacgctgt tacgatttca aaatgtgcaa tcgcttcacc gtcgcgtacg 1380 tattttcatgattgtctgcg ttctgtggtg cgtctggatt tgtctctcga cgtttctgat 1440 agccatgttccatcgacgat cctcgggaat gccagagtag attttcatga atccacaggc 1500 tgcggtgtccggacggcgaa gtctgctaca gtcccgagaa aacggctgag attcgcggga 1560 tcgtcaccaccatgacccat tcattgacac gccaggtcgt acacaacaaa ctgacgagct 1620 gcaactacaatccgtaagtc tcttcctcga gggccttaca gcctatggga aagtaagaca 1680 gagggacaaaacatcattaa aaaaaaagtc taatttcacg ttttgtaccc ccccttcccc 1740 tccgtgttgtaggttatacc tcgaagctga cgggcgaata cgctgcggca aagtgaacga 1800 caaggcgcagtacctgctgg gcgccgctgg cagcgttccc tatcgatgga tcaacctgga 1860 atacgacaagataacccgga tcgtgggcct ggatcagtac ctggagagcg ttaagaaaca 1920 caaacggctggatgtgtgcc gcgctaaaat gggctatatg ctgcagtgaa taataaa 1977 34 129 PRTHuman cytomegalovirus 34 Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys LeuCys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg Glu Thr Ala Glu LysAsn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp Ala Cys Ser Arg AlaLeu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu Gln Leu Val Asp LeuThr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly Leu Asp Asn Phe AspVal Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val Ser Leu Leu Ile SerAsp Phe Arg Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr Asn Lys Arg Thr ThrPhe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His Ala Arg Ser Leu GluPhe Ser Val Arg Leu Phe Ala 115 120 125 Asn 35 30 DNA ArtificialSequence oligonucleotide primer 35 cggcacacat ccagccgttt gtgtttctta 3036 30 DNA Artificial Sequence oligonucleotide primer 36 taacgctctccaggtactga tccaggccca 30 37 30 DNA Artificial Sequence oligonucleotideprimer 37 tcgtcagttt gttgtgtacg acctggcgtg 30 38 30 DNA ArtificialSequence oligonucloetide primer 38 tattggcctc ggtgaacgtc aatcgcacct 3039 30 DNA Artificial Sequence oligonucleotide primer 39 tgtgtcgggtgtggctgtct gtttgtctgt 30 40 30 DNA Artificial Sequence oligonucleotideprimer 40 tctgcttcgt caccactttc actgcctgct 30 41 30 DNA ArtificialSequence oligonucleotide primer 41 cgcagaagaa tgttgcgaat tcataaacgt 3042 30 DNA Artificial Sequence oligonulceotide primer 42 gctgcggtgtccggacggcg aagtctgcta 30 43 30 DNA Artificial Sequence oligonucleotideprimer 43 ccagctggca gattcccaaa ctaatgaaag 30 44 30 DNA ArtificialSequence oligonucleotide primer 44 ctttcggttc caactctttc cccgccccat 3045 30 DNA Artificial Sequence oligonucleotide primer 45 cacctcgcctatactatgtg tatgatgtct 30 46 30 DNA Artificial Sequence oligonucleotideprimer 46 ctctctttct cagtctgcaa catgcggctg 30 47 30 DNA ArtificialSequence oligonucleotide primer 47 gttgtccaag ccgtcgctcg catcgtagtg 3048 30 DNA Artificial Sequence oligonucleotide primer 48 cataataaagctctctttct cagtctgcaa 30 49 30 DNA Artificial Sequence oligonucleotideprimer 49 tatgatgtct cataataaag ctctctttct 30 50 390 DNA Humancytomegalovirus 50 atgcggctgt ctcgggtgtg gctgtctgtt tgtctgtgcgccgtggtgct gggtcagtgc 60 cagcgggaga ccgcagaaaa aaacgattat taccgagtaccgcattactg ggacgcgtgc 120 tctcgcgcgc tgcctgacca aacccgttac aagtatgtggaacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggc ttggacaactttgacgtgct caagagaatc 240 aacgtgaccg aggtgtcgtt gctcatcagc gactttatacgtcagaaccg tcgcggcggc 300 accaacaaaa ggaccacgtt caacgccgcc ggttcgctggcgcctcacgc ccggagcctc 360 gagttcagcg tgcggctctt tgccaactag 390 51 129PRT Human cytomegalovirus 51 Met Arg Leu Ser Arg Val Trp Leu Ser Val CysLeu Cys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg Glu Thr Ala GluLys Asn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp Ala Cys Ser ArgAla Leu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu Gln Leu Val AspLeu Thr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly Leu Asp Asn PheAsp Val Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val Ser Leu Leu IleSer Asp Phe Ile Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr Asn Lys Arg ThrThr Phe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His Ala Arg Ser LeuGlu Phe Ser Val Arg Leu Phe Ala 115 120 125 Asn 52 240 DNA Humancytomegalovirus 52 atgcggctgt gtcgggtgtg gctgtctgtt tgtctgtgcgccgtggtgct gggtcagtgc 60 cagcgggaga ccgcagaaaa aaacgattat taccgagtaccgcattactg ggacgcgtgc 120 tctcgcgcgc tgcctgacca aacccgttac aagtatgtggaacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggc ttggacaactttgacgtgct caagaggtga 240 53 79 PRT Human cytomegalovirus 53 Met Arg LeuCys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu GlyGln Cys Gln Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val ProHis Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg TyrLys Tyr Val Glu Gln Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr AspAla Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg 65 70 75 54 1977 DNAHuman cytomegalovirus 54 gtctgcaaca tgcggctgtg tcgggtgtgg ctgtctgtttgtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagac cgcagaaaaa aacgattattaccgagtacc gcattactgg 120 gacgcgtgct ctcgcgcgct gcctgaccaa acccgttacaagtatgtgga acagctcgtg 180 gacctcacgt tgaactacca ctacgatgcg agccacggcttggacaactt tgacgtgctc 240 aagaggtgag ggtacgcgct aaaggtgtat gacaacgggaaggtaagggc gaacgggtaa 300 cgggtaggta accgcatggg gtgtgaaatg acgttcggaacctgtgcttg cagaatcaac 360 gtgaccgagg tgtcgttgct catcagcgac tttagacgtcagaaccgtcg cggcggcacc 420 aacaaaagga ccacgttcaa cgccgccggt tcgctggcgcctcacgcccg gagcctcgag 480 ttcagcgtgc ggctctttgc caactagcct gcgtcacgggaaataatatg ctacggcttc 540 tgcttcgtca ccactttcac tgcctgcttc tgtgcgcggtttgggcaacg ccctgtctgg 600 cgtctccgtg gttcacgcta acggcgaacc agaatccgtccccgccatgg tctaaactga 660 cgtatcccaa accgcatgac gcggcgacgt tttactgtccttttctctat ccctcgcccc 720 cacggtcccc ctcgcaattc ccggggttcc agcgggtatcaacgggtccc gagtgtcgca 780 acgagaccct gtatctgctg tacaaccggg aaggccagaccttggtggag agaagctcca 840 cctgggtgaa aaaggtgatc tggtatctga gcggtcgcaatcagaccatc ctccaacgga 900 tgccccgaac ggcttcgaaa ccgagcgacg gaaacgtgcagatcagcgtg gaagacgcca 960 agatttttgg agcgcacatg gtgcccaagc agaccaagctgctacgtttc gtcgtcaacg 1020 atggcacacg ttatcagatg tgtgtgatga aactggagagctgggcccac gtcttccggg 1080 actacagcgt gtcttttcag gtgcgattga cgttcaccgaggccaataac cagacttaca 1140 ccttctgcac ccatcccaat ctcatcgttt gagcccgtcgcgcgcgcagg gaattttgaa 1200 aaccgcgcgt catgagtccc aaaaacctga cgccgttcttgacggcgttg tggctgctat 1260 tgggtcacag ccgcgtgccg cgggtacgcg cagaagaatgttgcgaattc ataaacgtca 1320 accacccgcc ggaacgctgt tacgatttca aaatgtgcaatcgcttcacc gtcgcgtacg 1380 tattttcatg attgtctgcg ttctgtggtg cgtctggatttgtctctcga cgtttctgat 1440 agccatgttc catcgacgat cctcgggaat gccagagtagattttcatga atccacaggc 1500 tgcggtgtcc ggacggcgaa gtctgctaca gtcccgagaaaacggctgag attcgcggga 1560 tcgtcaccac catgacccat tcattgacac gccaggtcgtacacaacaaa ctgacgagct 1620 gcaactacaa tccgtaagtc tcttcctcga gggccttacagcctatggga aagtaagaca 1680 gagggacaaa acatcattaa aaaaaaagtc taatttcacgttttgtaccc ccccttcccc 1740 tccgtgttgt aggttatacc tcgaagctga cgggcgaatacgctgcggca aagtgaacga 1800 caaggcgcag tacctgctgg gcgccgctgg cagcgttccctatcgatgga tcaacctgga 1860 atacgacaag ataacccgga tcgtgggcct ggatcagtacctggagagcg ttaagaaaca 1920 caaacggctg gatgtgtgcc gcgctaaaat gggctatatgctgcagtgaa taataaa 1977 55 1741 DNA Human cytomegalovirus 55 atgcggctgtctcgggtgtg gctgtctgtt tgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggagaccgcagaaaa aaacgattat taccgagtac cgcattactg ggacgcgtgc 120 tctcgcgcgctgcctgacca aacccgttac aagtatgtgg aacagctcgt ggacctcacg 180 ttgaactaccactacgatgc gagccacggc ttggacaact ttgacgtgct caagagaatc 240 aacgtgaccgaggtgtcgtt gctcatcagc gactttatac gtcagaaccg tcgcggcggc 300 accaacaaaaggaccacgtt caacgccgcc ggttcgctgg cgcctcacgc ccggagcctc 360 gagttcagcgtgcggctctt tgccaactag cctgcgtcac gggaaataat atgctacggc 420 ttctgcttcgtcaccacttt cactgcctgc ttctgtgcgc ggtttgggca acgccctgtc 480 tggcgtctccgtggttcacg ctaacggcga accagaatcc gtccccgcca tggtctaaac 540 tgacgtatcccaaaccgcat gacgcggcga cgttttactg tccttttctc tatccctcgc 600 ccccacggtccccctcgcaa ttcccggggt tccagcgggt atcaacgggt cccgagtgtc 660 gcaacgagaccctgtatctg ctgtacaacc gggaaggcca gaccttggtg gagagaagct 720 ccacctgggtgaaaaaggtg atctggtatc tgagcggtcg caatcagacc atcctccaac 780 ggatgccccgaacggcttcg aaaccgagcg acggaaacgt gcagatcagc gtggaagacg 840 ccaagatttttggagcgcac atggtgccca agcagaccaa gctgctacgt ttcgtcgcca 900 acgatggcacacgttatcag atgtgtgtga tgaaactgga gagctgggcc cacgtcttcc 960 gggactacagcgtgtctttt caggtgcgat tgacgttcac cgaggccaat aaccagactt 1020 acaccttctgcacccatccc aatctcatcg tttgagcccg tcgcgcgcgc agggaatttt 1080 gaaaaccgtgcgtcatgagt cccaaaaacc tgacgccgtt cttgacggcg ttgtggctgc 1140 tattgggtcacagccgcgtg ccgcgggtac gcgcagaaga atgttgcgaa ttcataaacg 1200 tcaaccacccgccggaacgc tgttacgatt tcaaaatgtg caatcgcttc accgtcgcgt 1260 acgtattttcatgattgtct gcgttctgtg gtgcgtctgg atctgtctct cgacgtttct 1320 gatagccatgttccatcgac gatcctcggg aatgccagag tagattttca tgaatccaca 1380 ggctgcggtgtccggacggc gaagtctgct acagtcccga gaaaacggct gagattcgcg 1440 ggatcgtcaccaccatgacc cattcattga cacgccaggt cgtacacaac aaactgacga 1500 actgcaactacaatccgtta tacctcgaag ctgacgggcg aatacgctgc ggcaaagtga 1560 acgacaaggcgcagtacctg ctgggcgccg ctggcagcgt tccctatcga tggatcaacc 1620 tggaatacgacaagataacc cggatcgtgg gcctggatca gtacctggag agcgttaaga 1680 aacacaaacggctggatgtg tgccgcgcta aaatgggcta tatgctgcag tgaataataa 1740 a 1741 56390 DNA Human cytomegalovirus 56 atgcggctgt ctcgggtgtg gctgtctgtttgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggaga ccgcagaaaa aaacgattattaccgagtac cgcattactg ggacgcgtgc 120 tctcgcgcgc tgcctgacca aacccgttacaagtatgtgg aacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggcttggacaact ttgacgtgct caagagaatc 240 aacgtgaccg aggtgtcgtt gctcatcagcgactttatac gtcagaaccg tcgcggcggc 300 accaacaaaa ggaccacgtt caacgccgccggttcgctgg cgcctcacgc ccggagcctc 360 gagttcagcg tgcggctctt tgccaactag390 57 129 PRT Human cytomegalovirus 57 Met Arg Leu Ser Arg Val Trp LeuSer Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg GluThr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp AlaCys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu GlnLeu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly LeuAsp Asn Phe Asp Val Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val SerLeu Leu Ile Ser Asp Phe Ile Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr AsnLys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His AlaArg Ser Leu Glu Phe Ser Val Arg Leu Phe Ala 115 120 125 Asn 58 1977 DNAHuman cytomegalovirus 58 gtctgcaaca tgcggctgtg tcgggtgtgg ctgtctgtttgtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagac cgcagaaaaa aacgattattaccgagtacc gcattactgg 120 gacgcgtgct ctcgcgcgct gcctgaccaa acccgttacaagtatgtgga acagctcgtg 180 gacctcacgt tgaactacca ctacgatgcg agccacggcttggacaactt tgacgtgctc 240 aagaggtgag ggtacgcgct aaaggtgtat gacaacgggaaggtaagggc gaacgggtaa 300 cgggtaggta accgcatggg gtgtgaaatg acgttcggaacctgtgcttg cagaatcaac 360 gtgaccgagg tgtcgttgct catcagcgac tttagacgtcagaaccgtcg cggcggcacc 420 aacaaaagga ccacgttcaa cgccgccggt tcgctggcgcctcacgcccg gagcctcgag 480 ttcagcgtgc ggctctttgc caactagcct gcgtcacgggaaataatatg ctacggcttc 540 tgcttcgtca ccactttcac tgcctgcttc tgtgcgcggtttgggcaacg ccctgtctgg 600 cgtctccgtg gttcacgcta acggcgaacc agaatccgtccccgccatgg tctaaactga 660 cgtatcccaa accgcatgac gcggcgacgt tttactgtccttttctctat ccctcgcccc 720 cacggtcccc ctcgcaattc ccggggttcc agcgggtatcaacgggtccc gagtgtcgca 780 acgagaccct gtatctgctg tacaaccggg aaggccagaccttggtggag agaagctcca 840 cctgggtgaa aaaggtgatc tggtatctga gcggtcgcaatcagaccatc ctccaacgga 900 tgccccgaac ggcttcgaaa ccgagcgacg gaaacgtgcagatcagcgtg gaagacgcca 960 agatttttgg agcgcacatg gtgcccaagc agaccaagctgctacgtttc gtcgtcaacg 1020 atggcacacg ttatcagatg tgtgtgatga aactggagagctgggcccac gtcttccggg 1080 actacagcgt gtcttttcag gtgcgattga cgttcaccgaggccaataac cagacttaca 1140 ccttctgcac ccatcccaat ctcatcgttt gagcccgtcgcgcgcgcagg gaattttgaa 1200 aaccgcgcgt catgagtccc aaaaacctga cgccgttcttgacggcgttg tggctgctat 1260 tgggtcacag ccgcgtgccg cgggtacgcg cagaagaatgttgcgaattc ataaacgtca 1320 accacccgcc ggaacgctgt tacgatttca aaatgtgcaatcgcttcacc gtcgcgtacg 1380 tattttcatg attgtctgcg ttctgtggtg cgtctggatttgtctctcga cgtttctgat 1440 agccatgttc catcgacgat cctcgggaat gccagagtagattttcatga atccacaggc 1500 tgcggtgtcc ggacggcgaa gtctgctaca gtcccgagaaaacggctgag attcgcggga 1560 tcgtcaccac catgacccat tcattgacac gccaggtcgtacacaacaaa ctgacgagct 1620 gcaactacaa tccgtaagtc tcttcctcga gggccttacagcctatggga aagtaagaca 1680 gagggacaaa acatcattaa aaaaaaagtc taatttcacgttttgtaccc ccccttcccc 1740 tccgtgttgt aggttatacc tcgaagctga cgggcgaatacgctgcggca aagtgaacga 1800 caaggcgcag tacctgctgg gcgccgctgg cagcgttccctatcgatgga tcaacctgga 1860 atacgacaag ataacccgga tcgtgggcct ggatcagtacctggagagcg ttaagaaaca 1920 caaacggctg gatgtgtgcc gcgctaaaat gggctatatgctgcagtgaa taataaa 1977 59 1849 DNA Human cytomegalovirus 59 atgcggctgtgtcgggtgtg gctgtctgtt tgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggagaccgcagaaaa aaacgattat taccgagtac cgcattactg ggacgcgtgc 120 tctcgcgcgctgcctgacca aacccgttac aagtatgtgg aacagctcgt ggacctcacg 180 ttgaactaccactacgatgc gagccacggc ttggacaact ttgacgtgct caagaggtga 240 gggtacgcgctaaaggtgta tgacaacggg aaggtaaggg cgaacgggta acgggtaggt 300 aaccgcatggggtgtgaaat gacgttcgga acctgtgctt gcagaatcaa cgtgaccgag 360 gtgtcgttgctcatcagcga ctttagacgt cagaaccgtc gcggcggcac caacaaaagg 420 accacgttcaacgccgccgg ttcgctggcg cctcacgccc ggagcctcga gttcagcgtg 480 cggctctttgccaactagcc tgcgtcacgg gaaataatat gctacggctt ctgcttcgtc 540 accactttcactgcctgctt ctgtgcgcgg tttgggcaac gccctgtctg gcgtctccgt 600 ggttcacgctaacggcgaac cagaatccgt ccccgccatg gtctaaactg acgtatccca 660 aaccgcatgacgcggcgacg ttttactgtc cttttctcta tccctcgccc ccacggtccc 720 cctcgcaattcccggggttc cagcgggtat caacgggtcc cgagtgtcgc aacgagaccc 780 tgtatctgctgtacaaccgg gaaggccaga ccttggtgga gagaagctcc acctgggtga 840 aaaaggtgatctggtatctg agcggtcgca atcagaccat cctccaacgg atgccccgaa 900 cggcttcgaaaccgagcgac ggaaacgtgc agatcagcgt ggaagacgcc aagatttttg 960 gagcgcacatggtgcccaag cagaccaagc tgctacgttt cgtcgtcaac gatggcacac 1020 gttatcagatgtgtgtgatg aaactggaga gctgggccca cgtcttccgg gactacagcg 1080 tgtcttttcaggtgcgattg acgttcaccg aggccgataa ccagacttac accttctgca 1140 cccatcccaatctcatcgtt tgagcccgtc gcgcgcgcag ggaattttga aaaccgcgcg 1200 tcatgagtcccaaaaacctg acgccgttct tgacggcgtt gtggctgcta ttgggtcaca 1260 gccgcgtgccgcgggtacgc gcagaagaat gttgcgaatt cataaacgtc aaccacccgc 1320 cggaacgctgttacgatttc aaaatgtgca atcgcttcac cgtcgcgtac gtattttcat 1380 gattgtctgcgttctgtggt gcgtctggat ctgtctctcg acgtttctga tagccatgtt 1440 ccatcgacgatcctcgggaa tgccagagta gattttcatg aatccacagg ctgcggtgtc 1500 cggacggcgaagtctgctac agtcccgaga aaacggctga gattcgcggg atcgtcacca 1560 ccatgacccattcattgaca cgccaggtcg tacacaacaa actgacgagc tgcaactaca 1620 atccgttatacctcgaagct gacgggcgaa tacgctgcgg caaagtgaac gacaaggcgc 1680 agtacctgctgggcgccgct ggcggcgttc cctatcgatg gatcaacctg gaatacgaca 1740 agatagcccggatcgtgggc ctggatcagt acctggagag cgttaagaaa cacaaacggc 1800 tggatgtgtgccgcgctaaa atgggctata tgctgcagtg aataataaa 1849 60 240 DNA Humancytomegalovirus 60 atgcggctgt gtcgggtgtg gctgtctgtt tgtctgtgcgccgtggtgct gggtcagtgc 60 cagcgggaga ccgcagaaaa aaacgattat taccgagtaccgcattactg ggacgcgtgc 120 tctcgcgcgc tgcctgacca aacccgttac aagtatgtggaacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggc ttggacaactttgacgtgct caagaggtga 240 61 79 PRT Human cytomegalovirus 61 Met Arg LeuCys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu GlyGln Cys Gln Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val ProHis Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg TyrLys Tyr Val Glu Gln Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr AspAla Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg 65 70 75 62 180 DNAHuman cytomegalovirus 62 atgagtccca aaaacctgac gccgttcttg acggcgttgtggctgctatt gggtcacagc 60 cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattcataaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccgtcgcgtacgt attttcatga 180 63 59 PRT Human cytomegalovirus 63 Met Ser ProLys Asn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 1 5 10 15 Leu GlyHis Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe IleAsn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys AsnArg Phe Thr Val Ala Tyr Val Phe Ser 50 55 64 515 DNA Humancytomegalovirus 64 atgagtccca aaaacctgac gccgttcttg acggcgttgtggctgctatt gggtcacagc 60 cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattcataaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccgtcgcactgcg gtgtccggac 180 ggcgaagtct gctacagtcc cgagaaacgg ctgagattcgcgggatcgtc accaccatga 240 cccattcatt gacacgccag gtcgtacaca acaaactgacgagctgcaac tacaatctgt 300 tatacctcga agctgacggg cgaatacgct gcggcaaagtgaacgacaag gcgcagtacc 360 tgctgggcgc cgctggcagc gttccctatc gatggatcaacctggaatac gacaagataa 420 cccggatcgt gggcctggat cagtacctgg agagcgttaagaaacacaaa cggctggatg 480 tgtgccgcgc taaaatgggc tatatgctgc agtga 515 65171 PRT Human cytomegalovirus 65 Met Ser Pro Lys Asn Leu Thr Pro Phe LeuThr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly His Ser Arg Val Pro Arg ValArg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn Val Asn His Pro Pro GluArg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn Arg Phe Thr Val Ala Leu ArgCys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser Pro Glu Lys Thr Ala Glu IleArg Gly Ile Val Thr Thr Met 65 70 75 80 Thr His Ser Leu Thr Arg Gln ValVal His Asn Lys Leu Thr Ser Cys 85 90 95 Asn Tyr Asn Leu Leu Tyr Leu GluAla Asp Gly Arg Ile Arg Cys Gly 100 105 110 Lys Val Asn Asp Lys Ala GlnTyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125 Pro Tyr Arg Trp Ile AsnLeu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135 140 Gly Leu Asp Gln TyrLeu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160 Val Cys ArgAla Lys Met Gly Tyr Met Leu Gln 165 170 66 804 DNA Human cytomegalovirus66 atgagtccca aaaacctgac gccgttcttg acggcgttgt ggctgctatt gggtcacagc 60cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccg tcgcgtacgt attttcatga 180ttgtctgcgt tctgtggtgc gtctggatct gtctctcgac gtttctgata gccatgttcc 240atcgacgatc ctcgggaatg ccagagtaga ttttcatgaa tccacaggct gcggtgtccg 300gacggcgaag tctgctacag tcccgagaaa acggctgaga ttcgcgggat cgtcaccacc 360atgacccatt cattgacacg ccaggtcgta cacaacaaac tgacgagctg caactacaat 420ccgtaagtct cttcctcgag ggccttacag cctatgggaa agtaagacag agggacaaaa 480catcattaaa aaaaaagtct aatttcacgt tttgtacccc cccttcccct ccgtgttgta 540ggttatacct cgaagctgac gggcgaatac gctgcggcaa agtgaacgac aaggcgcagt 600acctgctggg cgccgctggc ggcgttccct atcgatggat caacctggaa tacgacaaga 660tagcccggat cgtgggcctg gatcagtacc tggagagcgt taagaaacac aaacggctgg 720atgtgtgccg cgctaaaatg ggctatatgc tgcagtgaat aataaaatgt gtgtttgtcc 780gaaatacgcg ttttgagatt tctg 804 67 685 DNA Human cytomegalovirus 67atgagtccca aaaacctgac gccgttcttg acggcgttgt ggctgctatt gggtcacagc 60cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccg tcgcgtacgt attttcatga 180ttgtctgcgt tctgtggtgc gtctggatct gtctctcgac gtttctgata gccatgttcc 240atcgacgatc ctcgggaatg ccagagtaga ttttcatgaa tccacaggct gcggtgtccg 300gacggcgaag tctgctacag tcccgagaaa acggctgaga ttcgcgggat cgtcaccacc 360atgacccatt cattgacacg ccaggtcgta cacaacaaac tgacgagctg caactacaat 420ccgttatacc tcgaagctga cgggcgaata cgctgcggca aagtgaacga caaggcgcag 480tacctgctgg gcgccgctgg cggcgttccc tatcgatgga tcaacctgga atacgacaag 540atagcccgga tcgtgggcct ggatcagtac ctggagagcg ttaagaaaca caaacggctg 600gatgtgtgcc gcgctaaaat gggctatatg ctgcagtgaa taataaaatg tgtgtttgtc 660caaaaaaaaa aaaaaaaaaa aaaaa 685 68 180 DNA Human cytomegalovirus 68atgagtccca aaaacctgac gccgttcttg acggcgttgt ggctgctatt gggtcacagc 60cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccg tcgcgtacgt attttcatga 180 6959 PRT Human cytomegalovirus 69 Met Ser Pro Lys Asn Leu Thr Pro Phe LeuThr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly His Ser Arg Val Pro Arg ValArg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn Val Asn His Pro Pro GluArg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn Arg Phe Thr Val Ala Tyr ValPhe Ser 50 55 70 780 DNA Human cytomegalovirus 70 atgagtccca aaaacctgacgccgttcttg acggcgttgt ggctgctatt gggtcacagc 60 cgcgtgccgc gggtacgcgcagaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaaaatgtgcaat cgcttcaccg tcgcgtacgt atttttatga 180 ttgtctgcgt tctgtggtgcgtctggattt gtctctcgac gtttctgata gccatgttcc 240 atcgacgatc ctcgggaatgccagagtaga ttttcatgaa tccacaggct gcggtgtccg 300 gacggcgaag tctgctacagtcccgagaaa acggctgaga ttcgcgggat cgtcaccacc 360 atgacccatt cattgacacgccaggtcgta cacaacaaac tgacgagctg caactacaat 420 ccgtaagtct cttcctcgagggccttacag cctatgggaa agtaagacag agggacaaaa 480 catcattaaa aaaaaagtctaatttcacgt tttgtacccc cccttcccct ccgtgttgta 540 ggttatacct cgaagctgacgggcgaatac gctgcggcaa agtgaacgac aaggcgcagt 600 acctgctggg cgccgctggcagcgttccct atcgatggat caacctggaa tacgacaaga 660 taacccggat cgtgggcctggatcagtacc tggagagcgt taagaaacac aaacggctgg 720 atgtgtgccg cgctaaaatgggctatatgc tgcagtgaat aataaaatgt gtgtttgtcc 780 71 529 DNA Humancytomegalovirus 71 atgagtccca aaaacctgac gccgttcttg acggcgttgtggctgctatt gggtcacagc 60 cgcgtgccgc gggtacgcgc agaagaatgt tgcgaattcataaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccgtcgcgctgcg gtgtccggac 180 ggcgaagtct gctacagtcc cgagaaaacg gctgagattcgcgggatcgt caccaccatg 240 acccattcat tgacacgcca ggtcgtacac aacaaactgacgagctgcaa ctacaatccg 300 ttatacctcg aagctgacgg gcgaatacgc tgcggcaaagtgaacgacaa ggcgcagtac 360 ctgctgggcg ccgctggcag cgttccctat cgatggatcaacctggaata cgacaagata 420 acccggatcg tgggcctgga tcagtacctg gagagcgttaagaaacacaa acggctggat 480 gtgtgccgcg ctaaaatggg ctatatgctg cagtgaataataaaatgtg 529 72 515 DNA Human cytomegalovirus 72 atgagtccca aaaacctgacgccgttcttg acggcgttgt ggctgctatt gggtcacagc 60 cgcgtgccgc gggtacgcgcagaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaaaatgtgcaat cgcttcaccg tcgcactgcg gtgtccggac 180 ggcgaagtct gctacagtcccgagaaacgg ctgagattcg cgggatcgtc accaccatga 240 cccattcatt gacacgccaggtcgtacaca acaaactgac gagctgcaac tacaatctgt 300 tatacctcga agctgacgggcgaatacgct gcggcaaagt gaacgacaag gcgcagtacc 360 tgctgggcgc cgctggcagcgttccctatc gatggatcaa cctggaatac gacaagataa 420 cccggatcgt gggcctggatcagtacctgg agagcgttaa gaaacacaaa cggctggatg 480 tgtgccgcgc taaaatgggctatatgctgc agtga 515 73 171 PRT Human cytomegalovirus 73 Met Ser Pro LysAsn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly HisSer Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile AsnVal Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn ArgPhe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser ProGlu Lys Thr Ala Glu Ile Arg Gly Ile Val Thr Thr Met 65 70 75 80 Thr HisSer Leu Thr Arg Gln Val Val His Asn Lys Leu Thr Ser Cys 85 90 95 Asn TyrAsn Leu Leu Tyr Leu Glu Ala Asp Gly Arg Ile Arg Cys Gly 100 105 110 LysVal Asn Asp Lys Ala Gln Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125Pro Tyr Arg Trp Ile Asn Leu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135140 Gly Leu Asp Gln Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145150 155 160 Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gln 165 170 74 1977DNA Human cytomegalovirus 74 gtctgcaaca tgcggctgtg tcgggtgtgg ctgtctgtttgtctgtgcgc cgtggtgctg 60 ggtcagtgcc agcgggagac cgcagaaaaa aacgattattaccgagtacc gcattactgg 120 gacgcgtgct ctcgcgcgct gcctgaccaa acccgttacaagtatgtgga acagctcgtg 180 gacctcacgt tgaactacca ctacgatgcg agccacggcttggacaactt tgacgtgctc 240 aagaggtgag ggtacgcgct aaaggtgtat gacaacgggaaggtaagggc gaacgggtaa 300 cgggtaggta accgcatggg gtgtgaaatg acgttcggaacctgtgcttg cagaatcaac 360 gtgaccgagg tgtcgttgct catcagcgac tttagacgtcagaaccgtcg cggcggcacc 420 aacaaaagga ccacgttcaa cgccgccggt tcgctggcgcctcacgcccg gagcctcgag 480 ttcagcgtgc ggctctttgc caactagcct gcgtcacgggaaataatatg ctacggcttc 540 tgcttcgtca ccactttcac tgcctgcttc tgtgcgcggtttgggcaacg ccctgtctgg 600 cgtctccgtg gttcacgcta acggcgaacc agaatccgtccccgccatgg tctaaactga 660 cgtatcccaa accgcatgac gcggcgacgt tttactgtccttttctctat ccctcgcccc 720 cacggtcccc ctcgcaattc ccggggttcc agcgggtatcaacgggtccc gagtgtcgca 780 acgagaccct gtatctgctg tacaaccggg aaggccagaccttggtggag agaagctcca 840 cctgggtgaa aaaggtgatc tggtatctga gcggtcgcaatcagaccatc ctccaacgga 900 tgccccgaac ggcttcgaaa ccgagcgacg gaaacgtgcagatcagcgtg gaagacgcca 960 agatttttgg agcgcacatg gtgcccaagc agaccaagctgctacgtttc gtcgtcaacg 1020 atggcacacg ttatcagatg tgtgtgatga aactggagagctgggcccac gtcttccggg 1080 actacagcgt gtcttttcag gtgcgattga cgttcaccgaggccaataac cagacttaca 1140 ccttctgcac ccatcccaat ctcatcgttt gagcccgtcgcgcgcgcagg gaattttgaa 1200 aaccgcgcgt catgagtccc aaaaacctga cgccgttcttgacggcgttg tggctgctat 1260 tgggtcacag ccgcgtgccg cgggtacgcg cagaagaatgttgcgaattc ataaacgtca 1320 accacccgcc ggaacgctgt tacgatttca aaatgtgcaatcgcttcacc gtcgcgtacg 1380 tattttcatg attgtctgcg ttctgtggtg cgtctggatttgtctctcga cgtttctgat 1440 agccatgttc catcgacgat cctcgggaat gccagagtagattttcatga atccacaggc 1500 tgcggtgtcc ggacggcgaa gtctgctaca gtcccgagaaaacggctgag attcgcggga 1560 tcgtcaccac catgacccat tcattgacac gccaggtcgtacacaacaaa ctgacgagct 1620 gcaactacaa tccgtaagtc tcttcctcga gggccttacagcctatggga aagtaagaca 1680 gagggacaaa acatcattaa aaaaaaagtc taatttcacgttttgtaccc ccccttcccc 1740 tccgtgttgt aggttatacc tcgaagctga cgggcgaatacgctgcggca aagtgaacga 1800 caaggcgcag tacctgctgg gcgccgctgg cagcgttccctatcgatgga tcaacctgga 1860 atacgacaag ataacccgga tcgtgggcct ggatcagtacctggagagcg ttaagaaaca 1920 caaacggctg gatgtgtgcc gcgctaaaat gggctatatgctgcagtgaa taataaa 1977 75 1620 DNA Human cytomegalovirus 75 atgcggctgtgtcgggtgtg gctgtctgtt tgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggagaccgcagaaaa aaaaacgatt attaccgagt accgcattac tgggacgcgt 120 gctctcgcgcgctgcctgac caaacccgtt acaagtatgt ggaacagctc gtggacctca 180 cgttgaactaccactacgat gcgagccacg gcttggacaa ctttgacgtg ctcaagagaa 240 tcaacgtgaccgaggtgtcg ttgctcatca gcgactttag acgtcagaac cgtcgcggcg 300 gcaccaacaaaaggaccacg ttcaacgccg ccggttcgct ggcgcctcac gcccggagcc 360 tcgagttcagcgtgcggctc tttgccaact agcctgcgtc acgggaaata atatgctacg 420 gcttctgcttcgtcaccact ttcactgcct gcttctgtgc gcggtttggg caacgccctg 480 tctggcgtctccgtggttca cgctaacggc gaaccagaat ccgtccccgc catggtctaa 540 actgacgtatcccaaaccgc atgacgcggc gacgttttac tgtccttttc tctatccctc 600 gcccccacggtccccctcgc aattcccggg gttccagcgg gtattaacgg gtcccgagtg 660 tcgcaacgagaccctgtatc tgctgtacaa ccgggaaggc cagaccttgg tggagagaag 720 ctccacctgggtgaaaaagg tgatctggca tctgagcggt cgcaatcaga ccatcctcca 780 acggatgccccgaacggctt cgaaaccgag cgacggaaac gtgcagatca gcgtggaaga 840 cgccaagatttttggagcgc acatggtgcc caagcagacc aagctgctac gtttcgtcgc 900 caacgatggcacacgttatt agatgtgtgt gatgaaactg gagagctggg cccacgtctt 960 ccgggactacagcgtgtctt ttcaggtgcg attgacgttc accgaggcca ataaccagac 1020 ttacaccttctgcacccatc ccaatctcat cgtttgagcc cgtcgcgcgc gcagggaatt 1080 ttgaaaaccgcgcgtcatga gtcccaaaaa cctgacgccg ttcttgacgg cgttgtggct 1140 gctattgggtcacagccgcg tgccgcgggt acgcgcagaa gaatgttgcg aattcataaa 1200 cgtcaaccacccgccggaac gctgttacga tttcaaaatg tgcaatcgct tcaccgtcgc 1260 actgcggtgtccggacggcg aagtctgcta cagtcccgag aaaacggctg agattcgcgg 1320 gatcgtcaccaccatgaccc attcattgac acgccaggtc gtacacaaca aactgacgag 1380 ctgcaactacaatctgttat acctcgaagc tgacgggcga atacgctgcg gcaaagtgaa 1440 cgacaaggcgcagtacctgc tgggcgccgc tggcagcgtt ccctatcgat ggatcaacct 1500 ggaatacgacaagataaccc ggatcgtggg cctggatcag tacctggaga gcgttaagaa 1560 atacaaacggctggatgtgt gccgcgctaa aatgggctat atgctgcagt gaataataaa 1620 76 645 DNAHuman cytomegalovirus 76 atgctacggc ttctgcttcg tcaccacttt cactgcctgcttctgtgcgc ggtttgggca 60 acgccctgtc tggcgtctcc gtggttcacg ctaacggcgaaccagaatcc gtccccgcca 120 tggtctaaac tgacgtatcc caaaccgcat gacgcggcgacgttttactg tccttttctc 180 tatccctcgc ccccacggtc cccctcgcaa ttcccggggttccagcgggt atcaacgggt 240 cccgagtgtc gcaacgagac cctgtatctg ctgtacaaccgggaaggcca gaccttggtg 300 gagagaagct ccacctgggt gaaaaaggtg atctggtatctgagcggtcg caatcagacc 360 atcctccaac ggatgccccg aacggcttcg aaaccgagcgacggaaacgt gcagatcagc 420 gtggaagacg ccaagatttt tggagcgcac atggtgcccaagcagaccaa gctgctacgt 480 ttcgtcgtca acgatggcac acgttatcag atgtgtgtgatgaaactgga gagctgggcc 540 cacgtcttcc gggactacag cgtgtctttt caggtgcgattgacgttcac cgaggccaat 600 aaccagactt acaccttctg cacccatccc aatctcatcgtttga 645 77 214 PRT Human cytomegalovirus 77 Met Leu Arg Leu Leu LeuArg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala ThrPro Cys Leu Ala Ser Pro Trp Phe Thr Leu Thr 20 25 30 Ala Asn Gln Asn ProSer Pro Pro Trp Ser Lys Leu Thr Tyr Pro Lys 35 40 45 Pro His Asp Ala AlaThr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro SerGln Phe Pro Gly Phe Gln Arg Val Ser Thr Gly 65 70 75 80 Pro Glu Cys ArgAsn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu ValGlu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp 100 105 110 Tyr Leu SerGly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala SerLys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 LysIle Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe CysThr 195 200 205 His Pro Asn Leu Ile Val 210 78 214 PRT Humancytomegalovirus 78 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys LeuLeu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro TrpPhe Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys LeuThr Tyr Pro Lys 35 40 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe LeuTyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro Ser Gln Phe Pro Gly Phe Gln ArgVal Ser Thr Gly 65 70 75 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu LeuTyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp ValLys Lys Val Ile Trp 100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile LeuGln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn ValGln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met ValPro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp GlyThr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala HisVal Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr PheThr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro AsnLeu Ile Val 210 79 171 PRT Human cytomegalovirus 79 Met Ser Pro Lys AsnLeu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly His SerArg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn ValAsn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn Arg PheThr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser Pro GluLys Thr Ala Glu Ile Arg Gly Ile Val Thr Thr Met 65 70 75 80 Thr His SerLeu Thr Arg Gln Val Val His Asn Lys Leu Thr Ser Cys 85 90 95 Asn Tyr AsnPro Leu Tyr Leu Glu Ala Asp Gly Arg Ile Arg Cys Gly 100 105 110 Lys ValAsn Asp Lys Ala Gln Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125 ProTyr Arg Trp Ile Asn Leu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135 140Gly Leu Asp Gln Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150155 160 Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gln 165 170

1. Study of the genetic region UL131-128 which determines leukotropism,monocyte tropism, endothelial cell tropism in human cytomegalovirus(HCMV) in FIX-BAC and all HCMV laboratory and wild-type strains as wellas BAC-cloned HCMV strains such as (TowL-BAC, HB-5-BAC, TowS-BAC,TB40E-BAC, Phoebe-BAC, Powers-BAC, AD169-BAC) and their respectivereconstituted viruses.
 2. Study and synthesis of the newly identifiedviral transcripts running through the UL131-128 genetic region which areeither spliced or unspliced, sense or anti-sense and which are encodingnovel C×C, CC chemokines or other attachment, fusion and cell attractionfactors.
 3. Study and synthesis of the newly disclosed protein productsHCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 as well as other potential proteinsencoded by the UL132-UL128 and UL131-128 genetic region of FIX-BAC,TowL-BAC, HB-5-BAC, TowS-BAC, TB40E-BAC, Phoebe-BAC, Powers-BAC,AD169-BAC, their respective reconstituted viruses, wild-type andlaboratory strains.
 4. Production of monoclonal antibodies againstHCK-1, HCK-2, HCK-3, HCK-4 and HCK-5, synthesis of chemotherapeuticagents interfering with HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 (forexample small molecules, anti-sense RNA, siRNA).
 5. Construction andstudy of cell lines which express or secrete HCK-1, HCK-2, HCK-3, HCK-4and HCK-5.
 6. Study of tissue tropism and pathogenesis of HCMV in vitroand in vivo by constructing virus mutants which express HCK-1, HCK-2,HCK-3, HCK-4 and HCK-5 or the newly identified transcripts (95-3, 95-8,95-11, 128A, 128B) or other as yet unidentified transcripts of theUL132-128 or UL131-128 region.
 7. Study of the transcriptional andposttranscriptional regulatory mechanisms which regulate or modify theexpression of HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 or other UL132-128 orUL131-128 encoded chemokine and microfusion inducing factors regardingtissue tropism, pathogenesis of HCMV, other herpesviruses as well as DNAand RNA viruses.
 8. Expression of HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5or the newly identified transcripts (95-3, 95-8, 95-11, 128A, 128B) inhuman or animal cells particularly immune cells in order to study orinfluence trafficking of such cells.
 9. Use of the newly identifiedvirus encoded chemokines HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 or thenewly identified transcripts (95-3, 95-8,95-11,128A, 128B) ortherapeutic agents directed against them for therapy of virus induceddiseases, autoimmune disease, cancer, atherosclerosis, vasculitis,rheumatoid disease, gene therapy, vector development, vaccinedevelopment, study of trafficking and migration of leukocytes,monocytes, dendritic cells, natural killer cells, T-cells, B-cells,study of latency and reactivation of HCMV, induction or prevention ofapoptosis, activation or resistance of virally infected target cells toNK cells and T cells (CTLs).
 10. Study of HCK-1, HCK-2, HCK-3, HCK-4 andHCK-5 or the newly identified transcripts (95-3, 95-8, 95-11, 128A,128B) in connection with C×C and CC chemokine receptor mediated entry ofHCMV, other Herpesviruses, other DNA and RNA viruses (for example HIV)into target cells and study of cell adherence mechanisms.
 11. Structuralanalyses of HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 and potentially otherchemokine, cytokine adherence and microfusion factors encoded by theUL132-128 or UL131-128 genetic locus.
 12. Study of co-infection ortransfection of target cells (expressing HCK-1, HCK-2, HCK-3, HCK-4 andHCK-5) by HCMV and other DNA and RNA viruses, especially HIV virus. 13.Study of HCK-1, HCK-2, HCK-3, HCK-4 and HCK-5 or the newly identifiedtranscripts (95-3, 95-8, 95-11, 128A, 128B) in vivo, in vitro and inanimal models in connection with the development of vascular damage,development of arteriitis, vasculitis, arteriosclerosis and stenosis ofthe vessel wall and mechanisms of protection against such diseases.